Detection of Potato mop top virus and Tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay

Tobacco rattle virus (TRV) and Potato mop top virus (PMTV) are important di seases of potato that are difficult to diagnose reliably by visual symptoms . Effective control strategies rely on accurate diagnosis. This paper descr ibes the development of a multiplex assay for the detection of TRV and PMTV directly from potato tubers and leaves by polymerase chain reaction (PCR) combined with in-tube fluorescent product detection (TaqMan). This technolo gy obviates any post-PCR manipulations and has many advantages including re ducing contamination risks, eliminating the need for ethidium bromide stain ing, and removing the time and cost of gel running. The new assay also allo ws the replacement of the two separate tests (a TRV reverse-transcription-P CR and a PMTV enzyme-linked immunosorbent assay) currently used with a sing le-tube multiplex format. In addition to greatly simplifying the detection of these two viruses, the multiplex TaqMan assay was also shown to be more sensitive than either of the tests that it replaces, allowing 100- and 10,0 00-fold increases in sensitivity for TRV and PMTV detection, respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtai ned from a wide range of different cultivars and geographical locations, in cluding some samples in which existing tests failed to detect virus. The us e of an assay of this kind in routine diagnosis helps to speed up and strea mline the diagnostic laboratory; in addition, more reliable diagnosis shoul d help in the control of this damaging disease.